Ben-Artzi H, Shemesh J, Zeelon E, Amit B, Kleiman L, Gorecki M, Panet A
BioTechnology General Ltd. Rehovot, Israel.
Biochemistry. 1996 Aug 13;35(32):10549-57. doi: 10.1021/bi960439x.
The molecular events leading to the second template switch during reverse transcription of the HIV genome were studied in a defined in-vitro system. In order to investigate displacement of the tRNA(lys) primer from the primer binding site (PBS) of the viral genomic RNA, following DNA synthesis, we produced an HIV RNA/DNA substrate that resembles the intermediate reverse transcription complex formed prior to the second template switch. Partial tRNA(lys) primer displacement was observed during plus (+) strand DNA synthesis and during minus (-) strand DNA elongation. We found two determinants that may serve as a stop signal for (+) DNA strong stop synthesis, the A(m) at position 19 of the natural tRNA(lys) and the secondary structure at the PBS sequence. The later signal appears to constitute a stronger terminator in-vitro. The 3' end of the nascent (-) DNA strand prior to the second template switch was also determined. It was mapped to the U5-PBS junction at the site for the first endonucleolytic cut introduced by the RNase H activity of the HIV reverse transcriptase (RT). Thus, different signals dictate the arrest of (-) and (+) nascent DNA synthesis. These stop signals appear to be required for the subsequent second template switch. However, an excess of (-) DNA "acceptor" molecules, having a 18-base sequence complementary to the (+) DNA "donor" template, was required to demonstrate the actual template switch in the in-vitro system. Taken together these results indicate that the reverse transcriptase can catalyze all the steps leading to the second template switch and auxiliary viral proteins may act to enhance the efficiency of this step during the reverse transcription process.
在一个特定的体外系统中研究了导致HIV基因组逆转录过程中第二次模板转换的分子事件。为了研究DNA合成后tRNA(lys)引物从病毒基因组RNA的引物结合位点（PBS）上的置换情况，我们制备了一种HIV RNA/DNA底物，它类似于第二次模板转换之前形成的中间逆转录复合物。在正（+）链DNA合成和负（-）链DNA延伸过程中均观察到了部分tRNA(lys)引物的置换。我们发现了两个可能作为（+）DNA强终止合成终止信号的决定因素，即天然tRNA(lys)第19位的A(m)以及PBS序列处的二级结构。后一个信号在体外似乎构成了更强的终止子。还确定了第二次模板转换之前新生（-）DNA链的3'末端。它被定位到HIV逆转录酶（RT）的RNase H活性引入的第一个内切核酸酶切割位点处的U5-PBS连接处。因此，不同的信号决定了（-）和（+）新生DNA合成的终止。这些终止信号似乎是后续第二次模板转换所必需的。然而，在体外系统中需要过量的具有与（+）DNA“供体”模板互补的18碱基序列的（-）DNA“受体”分子来证明实际的模板转换。综合这些结果表明，逆转录酶可以催化导致第二次模板转换的所有步骤，并且辅助病毒蛋白可能在逆转录过程中起到提高这一步骤效率的作用。
